The overall goal of this Program Project is to develop improved vectors for gene delivery in vivo and to test their efficacy in disease models in animals. The Program Project centers on the helper-dependent adenovirus system (HD-Ad) first developed at Baylor. HD-Ads are Ads that lack all viral protein genes. During the last funding cycle we showed that HD-Ads are highly efficient in transferring gene in vivo. There is minimal toxicity and transgenes delivered by such vectors are expressed for >2 year in baboons and for the lifetime of the recipient mice (2.5 years). In this competing renewal application, Dr. Stefan Kochanek (University of Cologne), a leader in HD-Ads, has formally joined the Program. As Dr. Frank Graham, director of one of the projects directed in the ongoing Program, will be retiring at the termination of the current cycle, his project will be directed by Dr. Philip Ng, a Graham-trained expert in HD-Ad who joined Baylor in November 2001. The Program will continue to improve HD-Ads and treatment protocols and to further test these vectors for toxicity and efficacy in disease models in mice and nonhuman primates. In Project 1 (Chan/Beaudet) we will generate an improved version of chimeric cDNA/genomic LDL receptor (LDLR) HD-Ad, and a dual LDLR+apoAI transgene vector, that will be tested in LDLR-deficient mice and rhesus monkeys, and to test the efficacy and toxicity of a glucose 6 phosphatase (G6Pase) HD-Ad for the treatment of glycogen storage disease 1a (GSD-1a) in mice and dogs (using a GSD-1a dog colony at Duke University). In Project 2 (Beaudet/Chan) we will elucidate the pathogenesis of HD-Ad-induced thrombocytopenia, a significant side effect of these vectors, evaluate the safety and toxicity of HD-Ads in nonhuman primates, and examine the efficacy of skeletal muscle gene transfer of Factor IX and apoAI as an alternative tissue target for HD-Ads in Factor IX-deficient and wild-type mice and in hyperlipidemic baboons. Data from these experiments will support possible clinical trials of muscle-directed therapy of Factor IX deficiency. In Project 3 (Kochanek) we will develop improved procedures for high-yield production of RCA (recombinant Ad)-free HD-Ads and vectors with enhanced hepatic transcription, and new pretreatment protocols and vector modifications that improve liver targeting and reduce toxicity; lastly, we will also determine chromosomal integration frequencies of HD-Ads. In Project 4 (Ng) we will optimize procedures and scaling up methods for HD-Ad production with reduced helper contamination and develop reagents suitable for clinical applications. The projects are supported by an Administrative Core (Chan), a Vector Production Core (Oka, PI) and a Nonhuman Primate Core (K. D. Carey, at the Southwest Foundation for Biomedical Research at San Antonio). HD-Ads are highly promising gene transfer vectors. The improvements in HD-Ads and the preclinical experiments proposed in this application will pave the way towards the application of this technology to their possible use in the treatment of human disease in clinical trials.